KCNE1 and KCNE3 beta-subunits regulate membrane surface expression of Kv12.2 K(+) channels in vitro and form a tripartite complex in vivo.
KCNE1 and KCNE3 beta-subunits regulate membrane surface expression of Kv12.2 K(+) channels in vitro and form a tripartite complex in vivo.
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DOI：
10.1371/journal.pone.0006330
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发表时间：
2009-07-22
期刊：
影响因子：
3.7
通讯作者：
Jegla T
中科院分区：
文献类型：
--
作者：
Clancy SM;Chen B;Bertaso F;Mamet J;Jegla T
在神经元静息膜电位附近激活的电压门控钾通道是神经系统中重要的兴奋调节剂，但其功能多样性仍不清楚。例如，Kv12.2（ELK 2，KCNH 3）通道在大脑皮层和海马中高度表达，尽管它们最有可能有助于静息钾电导，但令人惊讶的是，对它们的功能或调节知之甚少。在这里，我们证明了辅助MinK（KCNE 1）和MiRP 2（KCNE 3）蛋白是Kv12.2通道功能的重要调节因子。通过siRNA沉默降低内源性KCNE 1或KCNE 3表达，显著增加爪蟾卵母细胞中约4倍的宏观Kv12.2电流。有趣的是，在KCNE 1和KCNE 3 siRNA的双重注射下观察到Kv12.2电流几乎增加了9倍，这表明了累加效应。与这些发现一致，KCNE 1和/或KCNE 3的过表达抑制Kv12.2电流。膜表面生物素化分析表明，KCNE 1和KCNE 3 siRNA显著增加Kv12.2的表面表达，而Kv12.2的总蛋白表达不受影响。KCNE 1和KCNE 3 siRNA使半数最大激活电压向更高的超极化电压偏移，表明KCNE 1和KCNE 3也可能抑制Kv12.2的激活门控。小鼠脑细胞膜的天然免疫共沉淀试验表明，KCNE 1和KCNE 3在体内同时与Kv12.2相互作用，表明存在新的KCNE 1-KCNE 3-Kv12.2通道三联复合物。这些数据共同表明，KCNE 1和KCNE 3直接与Kv12.2通道相互作用，以调节通道膜运输。
Voltage-gated potassium channels that activate near the neuronal resting membrane potential are important regulators of excitation in the nervous system, but their functional diversity is still not well understood. For instance, Kv12.2 (ELK2, KCNH3) channels are highly expressed in the cerebral cortex and hippocampus, and although they are most likely to contribute to resting potassium conductance, surprisingly little is known about their function or regulation. Here we demonstrate that the auxiliary MinK (KCNE1) and MiRP2 (KCNE3) proteins are important regulators of Kv12.2 channel function. Reduction of endogenous KCNE1 or KCNE3 expression by siRNA silencing, significantly increased macroscopic Kv12.2 currents in Xenopus oocytes by around 4-fold. Interestingly, an almost 9-fold increase in Kv12.2 currents was observed with the dual injection of KCNE1 and KCNE3 siRNA, suggesting an additive effect. Consistent with these findings, over-expression of KCNE1 and/or KCNE3 suppressed Kv12.2 currents. Membrane surface biotinylation assays showed that surface expression of Kv12.2 was significantly increased by KCNE1 and KCNE3 siRNA, whereas total protein expression of Kv12.2 was not affected. KCNE1 and KCNE3 siRNA shifted the voltages for half-maximal activation to more hyperpolarized voltages, indicating that KCNE1 and KCNE3 may also inhibit activation gating of Kv12.2. Native co-immunoprecipitation assays from mouse brain membranes imply that KCNE1 and KCNE3 interact with Kv12.2 simultaneously in vivo, suggesting the existence of novel KCNE1-KCNE3-Kv12.2 channel tripartite complexes. Together these data indicate that KCNE1 and KCNE3 interact directly with Kv12.2 channels to regulate channel membrane trafficking.
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